Dear All,
In order to understand the protein interactome of my gene of interest, I am planning to tag the endogenous locus of the gene with V5 epitope and to perform mass spectrometry-based proteomic analysis, followed by co-immunoprecipitation against V5 epitope.
Do you think that V5 epitope is much suitable for this approach?
What are methodologies that I can use to reduce the background and to increase the resolution of my results?
Will it be better to have a crosslinking step/ method for the procedure?
Thank you with warm regards
Hi Lahiru,
V5 tag should be suitable for proteomics, although generally it is advisable to include a second tag so you can perform tandem affinity enrichment of your bait. Common setups are FLAG-HA-bait. Depending on your bait, a single pulldown is not sufficient to reduce background levels.
Most co-IP experiments involve "native" conditions, but sometimes crosslinking can be the preferred approach to reduce background. In that case, generally it is advisable to not use tags such as FLAG which can be inactivated due to the formaldehyde. One potential solution is BioTAP-XL, seen in a paper I co-authored and attached (PMID: 25559106).
You may have to modify the BioTAP-XL protocol to fit your needs, for instance depending on where your protein of interest is localized in the cell. However, crosslinking is useful because it allows you to efficiently inactivate proteases/nucleases and to solubilize your cells/pellet more efficiently with sonication. Additionally the BioTAP tag has much higher affinity for the respective resins than V5 tag, so you can wash more stringently and reduce your background recovery of proteins.
Let me know if I can answer more questions.
Thanks,
Barry
Article BioTAP-XL: Cross-linking/Tandem Affinity Purification to Stu...
Hello Lahiru Chamara Weerasinghe Arachchige
I would advise using magnetic beads and cross-link your anti-V5 antibody to it using DMP. The use of magnetic beads over agarose will reduce your background significantly. And ensuring you have a bead-only control will help determine which proteins are non-specific.
Also, will you be able to V5 label your protein separately at the N- and C- terminals to see what effect that has?
Good luck!
Peter
Hi Barry Zee and Peter Allen Faull ,
Thank you very much for your kind and detailed answers and recommendations.
Since most of the published works used flag tag for high throughput MS analysis. We decided to go with the 3XFlag tag and co-immunoprecipitation with dynabeads followed by LC-MS/MS.
I am working with the Greb1l gene, which a novel gene and has so little information about it. It produced a big protein, which is about 210 kDa in size. Currently, we have no information about its interacting partners, protein complexes or functions. What we know is that mutations in this gene course renal agenesis in both humans and mice. So my plan is to tag the endogenous locus of this gene with the 3X Flag tag in mouse ESCs, in vitro differentiate them into the tissue I want, and perform MS analysis on 3X flag purified proteins to find out its direct interacting partners. I tried to predict its structure using Swissmodel, but could not get a significant structure. Therefore, we are planning to produce two mESC lines that tag either the N or the C terminal of the proteins and use products from both of them for MS analysis. Do you think that it is worth to do that?
Since formaldehyde is not compatible with the flag tag, my plan is to in vivo crosslink my samples using the DSG or DST. I chose these crosslinkers because they have a shorter spacer arm length.
I am planing my Co-IP in native conditions. Will the DMP and in vivo crosslinking affect my protein's interactions? Or will it make some artificial backgrounds?
To reduce the background, I am thinking to treat my protein extracts firstly with IgG coated dynabeads, and then with the antibody-treated dynabeads. Do you think it will be a good idea for this experiment?
I am very looking forward to having your answers
Thank you very much again
Cheers...
Chamara
Hi Chamara,
Generating one cell line with an N-term tagged bait and another cell line with a C-term tagged bait is a good idea since the tag itself may affect your protein function/localization depending on tag placement. You may also consider placing a GGSG-type linker between your bait and tag.
Treating your extracts with IgG beads as you described is one way to reduce non-specific binders. Some people also “block” their beads with non-Flag extract and use the beads for the actual IP with Flag extract. This step is probably not needed since you’re presumably doing competitive elution of your complexes.
Thanks,
Barry
Hi Barry Zee ,
Thank you very much for your advice.
What exactly did you mean by 'competitive elution'?
Do you have any advice about what methods are more suitable to elute protein complexes in their native conditions from the antibody/magnetic bead after Co-IP?
Thanks
Hi Chamara,
Competitive elution involves adding excess molar quantities of free peptide, for instance free FLAG peptide, to displace your bait-associated complex from the anti-FLAG beads.
Generally if your complex is bound to the bead after IP, such peptide competition is the gentlest way to elute your complex. Another method is to clone a protease cleavage sequence between your tag and your bait, and then incubate your complex-bead mixture after IP with the appropriate protease to release the complex into the supernatant.
Thanks,
Barry
HI Barry Zee ,
I am using 3XFlag tag, which contains an enterokinase cleavage site. Do you think that it is a good choice for complex elutions?
Thanks
Hi Chamara,
It's worth to compare 3X-FLAG peptide and enterokinase digestion in elution efficiency. You'll probably want to remove enterokinase after cleavage for your downstream experiments. One way to do that is to incubate your elutions with anti-enterokinase resin and collect the flowthrough.
With peptide competion, you can remove excess free 3X-FLAG peptide from your eluted complex with a spin concentrator at low molecular weight cutoff, dialysis membrane, or SDS-PAGE.
Thanks,
Barry